Post-translational modifications (PTMs) of histone proteins, such as acetylation, methylation, phosphorylation, and ubiquitylation, play essential roles in regulating chromatin dynamics and gene expression (Jenuwein and Allis, Science, 2001, 293(5532):1074-80). Combinations of different modifications on histone proteins, termed the ‘histone code’, extend the information potential and regulate the readout of the genetic code. In addition to histones it has been found that many PTMs occur on non-histone proteins. These PTMs regulate protein-protein interactions, stability, localization, and/or enzymatic activities of proteins (Sims and Reinberg, Nat Rev Mol Cell Biol., 2008, 9:815-20). Therefore PTMs on non-histone proteins (e.g. on transcription factors) can substantially alter protein function, extending the regulatory role of PTMs to multiple cellular pathways (Benayoun and Veitia, Trends Cell Biol., 2009, 19(5):189-97). Along with serine, threonine and tyrosine phosphorylation, lysine methylation also plays a critical role in cell function (Huang and Berger, Curr Opin Genet Dev, 2008, 18(2):152-8). The enzymes responsible for lysine methylation were initially found to target histones. Accumulating evidence confirmed that some of these enzymes are not completely histone specific, but rather have a broader spectrum of protein substrates and are therefore termed protein lysine methyltransferases (PKMTs) (Lanouette et al., Mol Syst Biol., 2014, 10:724). Misregulation of PKMTs has been reported in cancer cell lines as well as in cancer patients (Miremadi et al., Hum Mol Genet., 2007, 16 Spec No 1:R28-49; Kudithipudi and Jeltsch, Biochim Biophys Acta, 2014, 1846(2):366-379) Accordingly, lysine was shown to influence different pathways directly linked to oncogenic transformation, providing a rationale for the involvement of PKMTs in cancer and for developing inhibitors for therapeutic intervention (Mair et al., Trends Pharmacol Sci., 2014, 35(3):136-45; Wagner and Jung, Nat Biotechnol., 2012, 30(7):622-3).
In the present invention, inhibitors directed against the PKMT SET and MYND domain-containing protein 2 (SMYD2) are described. SMYD2 is a catalytic SET domain containing protein methyltransferase reported to monomethylate several lysine residues on histone and non-histone proteins. Initially SMYD2 was characterized to methylate H3 lysine 36 (Brown et al., Mol Cancer., 2006, 5:26) and lysine 4 when interacting with HSP90a (Abu-Farha et al., Mol Cell Proteomics, 2008, 7(3):560-722008). Methylation of histones by SMYD2 has been connected to increased transcription of genes involved in cell cycle regulation, chromatin remodeling, and transcriptional regulation (Abu-Farha et al., Mol Cell Proteomics, 2008, 7(3):560-722008). In addition to the function of SMYD2 in transcriptional regulation, several studies uncovered an important role of SMYD2 methylation activity on non-histone proteins closely connected to cancer.
For example, the p53 tumor suppressor gene is mutated in approximately 50% of human cancers and protein activity is frequently repressed in the non-mutated cases, indicating a central role of p53 in preventing tumorgenesis (Levine, Cell, 1997, 88(3):323-31). It has been demonstrated that the activity of p53 protein is inhibited by SMYD2 mediated posttranslational methylation at lysine 370 (K370) (Wu et al., Biochemistry, 2011, 50(29):6488-97; Huang et al., Nature, 2006, 444(7119):629-32;). The structural basis of p53 methylation by SMYD2 has been characterized by solving the crystal structure of a ternary complex with cofactor product S-adenosylhomocysteine and a p53 substrate peptide (Wang et al., J Biol Chem., 2011, 286(44):38725-37). Methylation at K370 reduces the DNA-binding efficiency of p53 and subsequently prevents the transcriptional activation of the tumor suppressive genes p21 and MDM2 (Huang et al., Nature, 2006, 444(7119):629-32). In the same study, a knockdown of SMYD2 and treatment with doxorubicin led to an increase in p53-mediated cell-cycle arrest and apoptosis in a cancer cell line model. In line with these observations, low SMYD2 gene expression was suggested as predictive marker of an improved response to doxorubicin and cyclophosphamide neoadjuvant chemotherapy in breast cancer patients (Barros Filho et al., Braz J Med Biol Res., 2010, 43(12):1225-31). Additionally, a regulatory role of SMYD2 on p53 activity was confirmed independently in heart biology. SMYD2 was characterized in a cardiomyocyte model to be a cardioprotective protein by methylating p53, thereby reducing p53 mediated apoptosis induction (Sajjad et al., Biochim Biophys Acta., 2014, 1843(11):2556-62). Therefore SMYD2 inhibitors may provide new therapeutic options for cancers with SMYD2-mediated inactivation of the p53 tumor suppressor.
Another study revealed an additional link to cancer chemotherapy by uncovering the SMYD2-dependent methylation of poly(ADP-Ribose) Polymerase-1 (PARP1). Methylation of PARP1 at lysine 528 (K528) positively regulated the poly(ADP-ribosyl)ation activity of oncogenic protein PARP1 in cancer cells (Piao et al., Neoplasia, 2014, 16(3):257-64). PARP1 is involved in the base excision pathway of DNA repair. Increased PARP1 activity is known as possible escape mechanism from apoptosis induction by DNA-damaging agents for cancer cells (Peralta-Leal et al., Clin Transl Oncol., 2008, 10(6):318-23). Knockdown of SMYD2 resulted in the reduction of PARP1 enzymatic activity, suggesting that SMYD2 inhibition could improve cancer chemotherapy efficacy (Piao et al., Neoplasia, 2014, 16(3):257-64).
The retinoblastoma protein (Rb) is a further important tumor suppressor protein regulated by SMYD2. Rb normally restricts DNA replication by preventing the progression from G1 to the replicative S phase of the cell division cycle, by binding to and inhibiting transcription factors of the E2F family (Weinberg, Cell, 1995, 81(3):323-30). SMYD2 methylates Rb at lysine 810 (K810) and 860 (K860). SMYD2 methylation of K810 enhances phosphorylation of Rb and its dissociation from E2F, which promotes abnormal cell cycle progression to S phase and proliferation in cancer (Cho et al., Neoplasia, 2012, 14(6):476-86) In line with these observations, it has been shown that knockdown of SMYD2 in an esophageal squamous cell carcinoma (ESCC) cell line overexpressing SMYD2 led to suppression of proliferation due to G1 arrest (Komatsu et al., Carcinogenesis, 2009, 30(7):1139-46). The HSP90 chaperone is another protein regulated by SMYD2. This protein is a crucial facilitator of oncogene addiction and cancer survival (Whitesell et al., Nat Rev Cancer., 2005, 5(10):761-72). Cancer cells are dependent on the HSP90 chaperone machinery to protect oncoproteins from misfolding and degradation. In a protein-protein interaction study, SMYD2 was identified as an interaction partner of HSP90 (Abu-Farha et al., J Mol Cell Biol., 2011, 3(5):301-8). Different studies revealed multiple sites of SMYD2 dependent HSP90 methylation at lysines 531 (K531) and 574 (K574) (Hamamoto et al., Cancer Lett., 2014, 351(1):126-33) and lysines K209 and K615 (Abu-Farha et al., J Mol Cell Biol., 2011, 3(5):301-8). Methylation was shown to be important for dimerization and chaperone complex stability. Initially HSP90 regulation by SMYD2 was described in normal muscle tissue maintenance (Donlin et al., Genes Dev., 2012, 26(2):114-9; Voelkel et al., Biochim Biophys Acta. 2013, 1833(4):812-22). Notably, an additional role of HSP90 methylation by SMYD2 in human carcinogenesis was reported (Hamamoto et al., Cancer Lett., 2014, 351(1):126-33). Knockdown of SMYD2 in cancer cell lines destabilized ERBB2 and CDK4 oncoproteins, and overexpression of methylated HSP90 accelerated proliferation of model cell lines indicating an additional cancer promoting role of SMYD2.
In the MCF7 breast cancer model it has been demonstrated that SMYD2-mediates estrogen receptor alpha (ERα) methylation at lysine 266 (K266). SMYD2 thereby also has a potential role in breast cancer by fine-tuning the functions of ERα and estrogen induced gene expression (Zhang et al., Proc Natl Acad Sci USA., 2013, 110(43):17284-9; Jiang et al., J Mol Biol. 2014, 426(20):3413-25). In cancers, several studies detected abnormally high expression of SMYD2. In a model of aggressive acute myeloid leukemia (AML) containing the MLL-AF9 fusion oncoprotein, SMYD2 expression was identified as part of a program of aberrant self-renewal genes linked to leukemia stem cells and poor prognosis (Zuber et al., Genes Dev., 2011, 25: 1628-1640). Different studies reported overexpression of SMYD2 in cancer cell lines as well as in ESCC, bladder carcinoma, gastric cancer and pediatric acute lymphoblastic leukemia patients (Komatsu et al., Carcinogenesis, 2009, 30(7):1139-46 and Br J Cancer, 2014, doi: 10.1038/bjc.2014.543; Cho et al., Neoplasia, 2012, 14(6):476-86; Sakamoto et al, 2014, 38(4):496-502). Notably higher SMYD2 expression in ESCC, gastric cancer, and acute lymphoblastic leukemia patients correlated with lower survival rate and was suggested to be a clinically relevant prognostic marker, further indicating an oncogenic role of SMYD2 (Komatsu et al., Carcinogenesis, 2009, 30(7):1139-46 and Br J Cancer, 2014, doi: 10.1038/bjc.2014.543; Sakamoto et al., Leuk Res., 2014, 38(4):496-502). In validation experiments in these reports, knockdown of SMYD2 in overexpressing ESCC, bladder and gastric cancer cell line models significantly reduced cell proliferation. One potential underlying explanation for higher SMYD2 expression in cancer patients was described for ESCC. The SMYD2 gene is localized in a genomic region around 1q32 q41 which has been found to be frequently amplified in ESCC cell lines and patients (Komatsu et al., Carcinogenesis, 2009, 30(7):1139-46; Pimkhaokham et al., Jpn J Cancer Res., 2000, 91(11):1126-33).
These studies indicate that the SMYD2 proteins play an essential role in various pathologies. It would therefore be desirable to find potent and selective inhibitors which prevent the SMYD2 methylation activity.